A strain of Pseudomonas meliticus ZTT7-4 and its application

Abstract

This invention relates to the field of microbial biocontrol, specifically to a strain of *Pseudomonas thaliana* ZTT7-4 and its applications. The *Pseudomonas thaliana* strain of this invention (… Pseudomonas mediterranea The sample has been registered and preserved at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20261056. Its average inhibition rate against pepper mild mottle virus reached 77.36%. Screening natural microorganisms with antagonistic effects against pepper mild mottle virus disease is in line with the development direction of green plant protection and has good application value.

Description

Technical Field

[0001] This invention relates to the field of microbial biological control, specifically to a strain of Pseudomonas thaliana ZTT7-4 and its applications. Background Technology

[0002] chili( Red pepper (L.) is the most widely planted vegetable crop in China. Pepper mild mottle virus (PMMoV) is one of the important viral diseases in pepper cultivation. It is a typical soil-borne and seed-borne virus that seriously affects the yield and quality of peppers, sweet peppers, and tobacco, among other Solanaceae crops. It has extremely strong environmental stability and contact infectivity. Early symptoms of PMMoV infection in peppers include chlorosis, mottling, and mosaic patterns on the leaves. Plant growth is slowed, peppers are small, and the peel develops chlorotic mottling. In severe cases, the peppers become deformed and necrotic, seriously affecting their marketability. Currently, there are no highly effective chemical pesticides for plant viruses. Production mainly relies on disease-resistant breeding and agricultural control, but these methods are time-consuming, costly, and prone to rapid viral mutation, often leading to loss of resistance. Currently, researchers are attempting to screen bacteria with antiviral activity from natural microorganisms. In research on antiviral microorganisms, *Pseudomonas* spp. are considered... Pseudomonas and Bacillus Bacillus Bacteria are currently the main subject of research. *Pseudomonas fluorescens* P. fluorescens and Pseudomonas putida P. putida It can reduce the incidence and severity of TSWV on tomatoes, while promoting tomato growth. The *Pseudomonas fluorescens* strain CZ-rif can effectively prevent TMV on tobacco, with a prevention rate of 45.85%, while its fermentation broth showed a 42.02% control effect against tobacco mosaic virus in the field. CN202410849285.X discloses that *Bacillus cereus* can inhibit pepper mild mottle virus, with an inhibition rate of 93.8%. Therefore, screening and developing microorganisms and their preparations with resistance to PMMoV, and developing safe, efficient, and environmentally friendly biological control resources, has significant industrial value. Summary of the Invention

[0003] To further control pepper mild mottle virus and obtain more useful biocontrol agents for pepper mild mottle virus, this invention, through extensive screening and testing, discovered a species of Pseudomonas thaliana (…). P. mediterranea This invention is completed because it has a significant inhibitory effect on PMMoV.

[0004] The technical solution of the present invention is as follows: In one aspect, the present invention provides a strain of Pseudomonas meliticum ZTT7-4 ( Pseudomonas MediterraneanZTT7-4), this strain has been deposited at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, on May 25, 2026, with accession number CCTCC NO: M 20261056.

[0005] In one aspect, the present invention provides a microbial inoculant containing the *Pseudomonas thaliana* ZTT7-4 strain described in this invention.

[0006] In this invention, the microbial agent is one of a liquid preparation, a lyophilized powder, or a solid fixative.

[0007] In one embodiment, the liquid formulation contains a *Pseudomonas thaliana* fermentation broth. Specifically, the fermentation broth containing the bacteria is obtained by large-scale fermentation of the *Pseudomonas thaliana* strain ZTT7-4 of this invention using suitable culture media and conditions.

[0008] In one embodiment, strain ZTT7-4 was inoculated into an optimized liquid culture medium (e.g., LB medium or a proprietary fermentation medium containing carbon, nitrogen, and inorganic salts), and deep liquid fermentation was carried out under suitable temperature (25–30°C), pH (6.5–7.5), and aerobic conditions for a fermentation period of 24–48 h, until the viable cell count reached ≥1×10⁻⁶. 9 The fermentation broth, with a concentration of CFU / mL, can be used directly as a liquid preparation or diluted for field spraying.

[0009] In one embodiment, the lyophilized powder is prepared by freeze-drying a fermentation broth containing *Pseudomonas thaliana* ZTT7-4 and a cryoprotectant. Common cryoprotectants include sucrose (5-15% by weight / volume), glycerol (1-5%), sodium alginate (0.5-2%), and albumin (0.1-1%), which can be used alone or in combination. The role of cryoprotectants is to ensure or reduce the mortality rate of the strain during freezing, thereby improving the activity of the bacteria. This formulation has significant advantages such as light weight, good stability during room temperature storage and transportation, and long shelf life (up to 12-18 months), making it suitable for commercial promotion.

[0010] In one embodiment, the solid fixative is obtained by immobilizing a fermentation broth containing *Pseudomonas thaliana* ZTT7-4 with a carrier. Commonly used carriers include inorganic carriers such as activated carbon, diatomaceous earth, porous ceramics, zeolite, perlite, and some organic synthetic polymer gel carriers (polyvinyl alcohol, calcium alginate gel, polyurethane foam). In practice, the fermentation broth and carrier can be mixed at a ratio of 1:0.5 to 1:5 (volume / mass ratio), and then dried at low temperature or naturally air-dried to allow the bacteria to firmly adhere to the surface or internal pores of the carrier. This formulation is convenient for rhizosphere application, soil mixing, or preparation as slow-release granules, making it suitable for preventative basal application.

[0011] In one aspect, the present invention discloses a method for preventing and controlling pepper mild mottle virus, wherein *Pseudomonas thaliana* ZTT7-4 is applied to subjects infected with pepper mild mottle virus. Preferably, the subjects are peppers, sweet peppers, or tobacco.

[0012] In one embodiment, the method for preventing and controlling pepper mild mottle virus according to the present invention includes spraying, spraying, and application in combination with fertilizers or pesticides to the target crop.

[0013] In one aspect, this invention provides the use of *Pseudomonas meliticus* ZTT7-4 in the control of pepper mild mottle virus (PMMoV). This strain exhibits significant inhibitory effects on PMMoV, effectively reducing the virus infection rate in pepper plants, alleviating viral disease symptoms, and improving pepper yield and quality.

[0014] Those skilled in the art will understand that, in this invention, "Pseudomonas meliticum ZTT7-4", " P. Mediterranean ZTT7-4” Pseudomonas mediterranea "ZTT7-4" and "ZTT7-4 strain" both refer to the *Pseudomonas thaliana* strain with accession number CCTCC NO: M 20261056 in this invention.

[0015] Beneficial effects This invention obtained a Mediterranean Pseudomonas strain ZTT7-4 that can inhibit pepper mild mottle virus through soil screening. Testing showed that strain ZTT7-4 achieved an average inhibition rate of 77.36% against pepper mild mottle virus. This provides a new method for controlling pepper mild mottle virus in production. Screening and utilizing natural microorganisms for plant virus control can promote the construction and innovation of a green prevention and control system for plant viral diseases, providing strong support for promoting green agriculture and achieving sustainable agricultural development. Attached Figure Description

[0016] Figure 1 The Mediterranean pseudomonas strain ZTT7-4 obtained by screening in this invention ( P. mediterranea ZTT7-4) growth status on the surface of NA medium.

[0017] Figure 2 The inhibitory effect of the Mediterranean Pseudomonas strain ZTT7-4 disclosed in this invention on PMMoV is shown in the figure. Figure 2 In the middle group A, the left half of the leaf was treated with liquid NA medium mixed with PMMoV as the control group; the right half of the leaf was treated with ZTT7-4 bacterial suspension mixed with PMMoV as the treatment group. Figure 2In the middle group (B), the left half of the leaf was treated with a mixture of sterile distilled water and PMMoV as the control group; the right half of the leaf was treated with a mixture of Ningnanmycin and PMMoV as the treatment group. Detailed Implementation

[0018] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. Unless otherwise specified, the equipment and reagents used in the embodiments and experimental examples are commercially available. Unless otherwise stated, all reagents used in this invention are analytical grade reagents. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention.

[0019] Example 1: Screening and Identification of Strains (1) Sampling: In the chili fields where viral diseases have broken out, select healthy chili plants and take 10-15 cm of rhizosphere soil.

[0020] (2) Take 10 g of soil sample, add 50 mL of sterile water, and shake thoroughly with glass beads until no large particles remain. Let stand at room temperature for 30 min. Take 100 μL of the supernatant and dilute it at a ratio of 1:10 (e.g., mix 1 mL of the original solution with 9 mL of physiological saline) to prepare 10 g of the original solution. - ¹Diluent. Continue with 10-fold serial dilutions (10... - ²、10 - (³ etc.), for later use.

[0021] (3) Take 100 μL of each dilution and spread it evenly on the surface of NA nutrient agar plates (beef extract 3.0 g / L, sucrose 10.0 g / L, peptone 5.0 g / L, yeast extract 1.0 g / L, agar 15.0 g / L, pH=7.1) using a sterile pipette. Gently tap the surface of the plate with sterile glass beads to distribute the bacterial solution evenly. Invert the plate and place it in a constant temperature incubator at 28℃ for 24-48 h.

[0022] (4) Single colony picking and purification: Observe the colonies, and after cultivation, pick single colonies with different morphologies (such as shape, edge regularity, colony color, etc.). Use a sterile inoculation loop to pick up a single colony and streak it on a new NA nutrient agar plate for isolation. Incubate at 28℃ with the plate inverted, repeating 2-3 times until a pure strain is obtained. The pure strain can be stored for a long time at -80℃ in 25% glycerol NA medium or used as a test strain for the half-leaf necrotic spot test.

[0023] (5) Screening: Single colony bacteria were selected and cultured in 100 mL Erlenmeyer flasks containing 20 mL of liquid NA medium (beef extract 3.0 g / L, sucrose 10.0 g / L, peptone 5.0 g / L, yeast extract 1.0 g / L, pH=7.1) at 28℃ and 200 rpm for 12-24 h with shaking to ensure a sufficiently high bacterial concentration, which was then used as a reserve inoculum for fermentation. The PMMoV necrotic host, *Nicotiana scabra* (*Nicotiana scabra*), was selected. Nicotiana tabacum The half-leaf method of inoculation was used to determine the inhibition rate of PMMoV infection by different strains of *S. samsun* var. *Samsun* using the allergic necrosis response of *S. samsun* to PMMoV.

[0024] (6) Identification: The strain ZTT7-4, which had the highest virus inhibition rate obtained in step (5), was selected and identified using conventional microbial identification methods such as colony growth and morphology observation. The ZTT7-4 strain obtained in this screening formed round, opaque, pale yellow, smooth-protruding, neatly edged, and sticky colonies on NA medium, and was Gram-negative. Pathogen DNA was extracted using the Omega Bacterial Genomic DNA Purification Kit (D3350-01). Based on the gene encoding the α subunit of RNA polymerase... rpoA Gene-specific primers rpoA-F (5'-ATGCAGAGTTCGGTAAATGAGTTCCTGACC-3', SEQ ID No. 2) and rpoA-R (5'-TCAGGCAGTGGCCTTGTCGTCTTTCTT-3', SEQ ID No. 3) were designed. PCR amplification was performed using TOYOBOKOD FX (KFX-101) under the following conditions: 94℃ pre-denaturation for 2 min; 98℃ denaturation for 10 s; 58℃ annealing for 30 s; 68℃ extension for 1 min 30 s, 30 cycles; and 68℃ extension for 10 min. The PCR products were recovered using an Omega gel recovery kit (D3350-01), then ligated, transformed, and sequenced using a Hieff Clone Zero TOP0-TACloning Kit (10907ES). The sequencing results are shown in SEQ ID No. 1. P. mediterranea of rpoA The gene similarity (Genbank accession number: CP102176.1) reached 99.60%. This bacterium was identified as... P. mediterranea It was named Pseudomonas thaliana ZTT7-4. This strain has been deposited at the China Center for Type Culture Collection (CCTCC) at Wuhan University, Wuhan, China, on May 25, 2026, with accession number CCTCC NO: M 20261056.

[0025] Example 2: Comparison of PMMoV inhibitory effects of Pseudomonas thaliana ZTT7-4 Pseudomonas thalassemia ZTT7-4 ( P. mediterranea Colonies of ZTT7-4 were fermented to obtain a bacterial solution. Single colonies of ZTT7-4 were picked and cultured in liquid NA medium at 28℃ and 200 rpm for 12-24 h. The OD of the bacterial solution was measured with a spectrophotometer until the bacterial solution was oxidized. 600 The target concentration was 0.8. Healthy, uniformly growing tobacco plants with 4-6 leaves were selected. Fully infected young leaves of PMMoV were used as the virus source, ground and filtered with phosphate buffer at a 1:10 (mass-to-volume) ratio, and used as the virus test sample. An equal volume of the virus test sample was mixed with liquid NA medium, and after 30 min, the sample was rubbed onto the left half of the leaf with necrotic spots as the control group. An equal volume of ZTT7-4 bacterial suspension was mixed with the virus test sample, and after 30 min, the sample was rubbed onto the right half of the leaf with necrotic spots as the treatment group. Ningnanmycin was used as a positive control. Following the same method, Ningnanmycin was mixed evenly with the virus test sample and applied to the right half of the leaf as the treatment group, while the left half of the leaf was not evenly coated with the bacterial suspension. A mixture of distilled water and the virus test sample was used as the control group. After 48 h, when necrotic spots appeared on the leaf surface, the number of necrotic spots on the left and right halves of the leaf was recorded. The experiment was repeated three times, with three leaves inoculated per tobacco plant. The results are as follows: Figure 2 As shown in Table 1, the number of necrotic spots on the left and right halves of the leaf and the inhibition rate are as follows: ; Table 1: Comparison of the inhibitory effects of different treatments on PMMoV Inhibition rate (%) = [(number of necrotic spots in control group - number of necrotic spots in treatment group) / number of necrotic spots in control group] × 100%, see Table 1. The average inhibition rate was used for evaluation. Strain ZTT7-4 had a significant antagonistic effect on PMMoV. Three repeated experiments showed that ZTT7-4 had an average inhibition rate of 77.36% against PMMoV, while ningnanmycin had an average inhibition rate of 40.16% against PMMoV. The inhibitory effect of Pseudomonas thaliana ZTT7-4 obtained by screening and comparison in this invention on PMMoV was significantly higher than that of the chemical pesticide ningnanmycin.

[0026] The above description, in conjunction with specific embodiments, provides a further detailed explanation of the present invention. It should not be construed that the specific implementation of the present invention is limited to these descriptions. For those skilled in the art, several simple deductions or substitutions can be made without departing from the concept of the present invention, and all such deductions or substitutions should be considered to fall within the scope of protection defined by the claims submitted herein.

Claims

1. A strain of Pseudomonas meliticus ZTT7-4, characterized in that, The strain is deposited at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, on May 25, 2026, with accession number CCTCCNO: M 20261056.

2. A microbial inoculant, characterized in that, The bacterial agent contains the Mediterranean Pseudomonas ZTT7-4 strain as described in claim 1.

3. The microbial agent according to claim 2, characterized in that, The bacterial agent is one of the following: liquid preparation, lyophilized powder, or solid fixative.

4. The microbial agent according to claim 3, characterized in that, The liquid preparation contains fermentation broth of Pseudomonas thaliana ZTT7-4.

5. The microbial agent according to claim 3, characterized in that, The freeze-dried powder was prepared by freeze-drying fermentation broth containing Pseudomonas thaliana ZTT7-4 and a freeze-drying protectant.

6. The microbial agent according to claim 3, characterized in that, The solid fixative is obtained by immobilizing and combining fermentation broth containing Pseudomonas thaliana ZTT7-4 with a carrier.

7. A method for preventing and controlling pepper mild mottle virus, characterized in that, The Mediterranean Pseudomonas ZTT7-4 strain as described in claim 1 was administered to subjects infected with pepper mild mottle virus.

8. The method according to claim 7, characterized in that, The object is a chili pepper, bell pepper, or tobacco.

9. The method according to claim 7, characterized in that, The methods include spraying, spraying, and applying to the target crop in combination with fertilizers or pesticides.

10. The use of the Mediterranean Pseudomonas ZTT7-4 according to claim 1 in the prevention and control of pepper mild mottle virus.

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